P-14 OPTIMIZATION OF MOLECULAR METHODS FOR SARS-CoV-2 QUALITATIVE DETECTION AND GENOTYPING IN RESPIRATORY SPECIMENS FROM PATIENTS WITH LIVER DISEASE

oleh: Vanessa Duarte da Costa, Alanna Calheiros Santos, Lucas Limas da Silva, Wilian Jean Wiggers, Claudia Alexandra Pontes Ivantes, Danielle Malta Lima, Jeová Keny Baima Colares, Deusilene Souza Vieira Dallacqua, Ana Rita Coimbra Motta-Castro, Vanessa Salete de Paula, Alberto Martín Rivera Dávila, Priscilla Pollo-Flores, Lia Laura Lewis-Ximenez, Livia Melo Villar

Format: Article
Diterbitkan: Elsevier 2023-03-01

Deskripsi

Introduction and Objectives: SARS-CoV-2 active infection diagnosis is currently performed through RT-qPCR. Despite the fact that PCR-based assays can provide results relatively fast, these techniques require capable professionals, specific equipment and adequate infrastructure. In order to facilitate COVID-19 diagnosis in remote areas, an alternative to RT-qPCR would be loop-mediated isothermal (RT-LAMP) amplification. SARS-CoV-2 variant genotyping through high-throughput sequencing (HTS) allows SARS-CoV-2 genomic surveillance, especially for patients with a higher vulnerability. This study aimed to optimize RT-LAMP and HTS methods for SARS-CoV-2 RNA detection and genotyping, respectively, in respiratory samples from patients with liver disease. Materials and Methods: A total of 142 respiratory secretions were obtained from individuals with SARS-CoV-2 RNA detectable by RT-qPCR (N1 Ct ≤ 30), divided into groups with (n=18) or without (n=124) liver disease. The study also enrolled 55 individuals who had SARS-CoV-2 RNA undetectable at RT-qPCR. For RT-LAMP methodology, primers were used for ORF1 gene amplification. As for HTS genotyping, the steps of cDNA synthesis, complete SARS-CoV-2 genome PCR amplification, preparation of genomic libraries and sequencing in MinION device were performed for 26 swab samples. Results: Samples with viral RNA detectable by RT-qPCR had a mean Ct value of 24.3 ± 3.75. Referring to RT-LAMP, it was observed a sensitivity of 71.1% (101/142). When considering RT-qPCR mean Ct value, RT-LAMP sensitivity was 88.9% (16/18), associated with a mean Ct of 23.3 ± 3.5 for patients with COVID and hepatitis. A specificity of 100% (55/55) was observed since all negative swabs tested by RT-qPCR were negative at RT-LAMP. Through sequencing by MinION, SARS-CoV-2 lineages gamma (7/26; 27%), zeta (1/26; 3.9%), delta (6/26; 23%) and omicron (12/26; 46.1%) were genotyped and detected by RT-LAMP. Conclusions: RT-LAMP demonstrated high sensitivity for molecular detection of SARS-CoV-2 RNA for patients with high viral load. Besides, RT-LAMP was capable of detecting all SARS-CoV-2 lineages genotyped by MinION in both groups.